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J. Biol. Chem., Vol. 283, Issue 23, 16178-16186, June 6, 2008

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ATP-binding Cassette A1-mediated Lipidation of Apolipoprotein A-I Occurs at the Plasma Membrane and Not in the Endocytic Compartments^*

From the Ottawa Health Research Institute, and Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ontario K1Y 4E9, Canada

ATP-binding cassette transporter (ABC) A1 is required for the lipidation of apolipoprotein A-I to generate high density lipoprotein (HDL). This process is proposed to occur through a retro-endocytosis pathway in which apoA-I internalizes with ABCA1 and generates HDL from the endosomal compartments before resecretion. The aim of this study was to determine the route of apoA-I endocytosis and whether endocytosis contributes to HDL biogenesis. Using confocal microscopy, we found that internalized apoA-I only transiently colocalized with transferrin, a retro-endocytosis marker. Instead, apoA-I perfectly colocalized with a bulk phase uptake marker (fluorescein isothiocyanate-dextran) and, at later time points, with LysoTracker in several cell models including macrophages, fibroblasts, and baby hamster kidney cells. ABCA1 colocalized poorly with internalized apoA-I. To determine the contribution of internalized apoA-I to HDL biogenesis, we specifically removed apoA-I from the cell surface and analyzed the fate of internalized apoA-I. We found that 23% of cell-associated apoA-I was internalized at steady state. Of internalized apoA-I, only 20% was converted to HDL, and the rest was degraded, consistent with a lysosomal destination. We also found that apoA-I was released approximately five times faster from the plasma membrane than from the intracellular compartments. From these kinetic parameters, we estimated that 5.6% of apoA-I that interacts with cells is degraded and that internalized apoA-I contributes to 1.4% of total HDL production. We also found that blocking endocytosis with sucrose or cytochalasin D did not decrease cholesterol efflux or HDL biogenesis. We therefore conclude that the plasma membrane is the main platform where ABCA1-mediated lipidation of apoA-I occurs.

Received for publication, November 26, 2007 , and in revised form, March 5, 2008.

^* This work was presented at the American Heart Association Conference held in Orlando, FL, on November 7th 2007. This work was supported by grants from the Canadian Institutes of Health Research, the Canada Innovation Foundation, and the Heart & Stroke Foundation of Canada. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. S1-S4.

¹ Supported by a postdoctoral fellowship from the Canadian Institutes

² Supported by an Ontario Graduate Scholarship.

³ To whom correspondence should be addressed. E-mail: xzha{at}ohri.ca.

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